Prevention and Reduction of Blood Loss

ABSTRACT

Methods are described for preventing or reducing ischemia and/or systemic inflammatory response in a patient such as perioperative blood loss and/or systemic inflammatory response in a patient subjected to cardiothoracic surgery, e.g. coronary artery bypass grafting and other surgical procedures, especially when such procedures involve extra-corporeal circulation, such as cardiopulmonary bypass.

RELATED APPLICATION

This application claims the benefit of U.S. application Ser. No.11/322,960, filed Dec. 30, 2005, which claims the benefit from U.S.application Ser. No. 10/456,986, filed Jun. 6, 2003, which claims thebenefit from U.S. Provisional Application No. 60/387,239, filed Jun. 7,2002, and U.S. Provisional Application No. 60/407,003, filed Aug. 28,2002.

The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Proteases are involved in a broad range of biological pathways. Inparticular, serine proteases such as kallikrein, plasmin, elastase,urokinase plasminogen activator, thrombin, human lipoprotein-associatedcoagulation inhibitor, and coagulation factors such as factors VIIa,IXa, Xa, XIa, and XIIa have been implicated in pathways affecting bloodflow, e.g., general and focal ischemia, tumor invasion, fibrinolysis,perioperative blood loss, and inflammation. Inhibitors of specificserine proteases, therefore, have received attention as potential drugtargets for various ischemic maladies.

One such inhibitor, aprotinin (also called bovine pancreatic trypsininhibitor or BPTI), obtained from bovine lung, has been approved in theUnited States for prophylactic use in reducing perioperative blood lossand the need for transfusion in patients undergoing cardiopulmonarybypass (CPB), e.g., in the course of a coronary artery bypass graftingprocedure. Aprotinin is commercially available under the trade nameTRASYLOL.RTM. (Bayer Corporation Pharmaceutical Division, West Haven,Conn.) and was previously approved for use to treat pancreatitis. Theeffectiveness of aprotinin is associated with its relativelynon-specific abilities to inhibit a variety of serine proteases,including plasma kallikrein and plasmin. These proteases are importantin a number of pathways of the contact activation system (CAS).

CAS is initially activated when whole blood contacts the surface offoreign substrates (e.g., kaolin, glass, dextran sulfate, or damagedbone surfaces). Kallikrein, a serine protease, is a plasma enzyme thatinitiates the CAS cascade leading to activation of neutrophils, plasmin,coagulation, and various kinins. Kallikrein is secreted as a zymogen(pre-kallikrein) that circulates as an inactive molecule until activatedby a proteolytic event early in the contact activation cascade. Clearly,specific inhibition of kallikrein would be a very attractive approach tocontrol blood loss associated with CPB and the onset of systemicinflammatory response (SIR) as would be encountered during, for example,various invasive surgical procedures.

Despite being the only licensed compound for preventing perioperativeblood loss in CPB for coronary artery bypass grafting (CABG) procedures,aprotinin is not as widely used as would be expected. There are seriousconcerns regarding the use of this bovine polypeptide in patients whorequire CPB, and in particular the use of this compound in CABGprocedures. Aprotinin is not specific for kallikrein, but interacts withadditional enzymes (e.g., plasmin) in multiple pathways. Thus, themechanism of action of aprotinin is largely speculative, and the lack ofprecise understanding of what is affected during aprotinin treatmentproduces the risk of complications during treatment. One frequentlycited complication is uncontrolled thrombosis, due to aprotinin'sactions upon the fibrinolytic pathway. There is concern not only oversuch hyperacute events as major vessel thrombosis in the perioperativeperiod, but also over graft patency after the CABG procedure.Furthermore, as a naturally occurring protein obtained from bovine lung,administration of aprotinin in humans can elicit severe hypersensitivityor anaphylactic or anaphylactoid reactions after the first and, moreoften, after repeat administration to patients. This is particularly ofconcern in the large number of patients who have repeat CABG procedures.In addition, there is an increasing public concern regarding use ofmaterial derived from bovine sources as a potential vector for thetransmission of bovine spongiform encephalopathy to humans.

These concerns make clear that a need remains for more effective andmore specific means and methods for preventing or reducing perioperativeblood loss and the onset of SIR in a patient subjected to surgeryresulting in activation of the CAS, such as CABG procedures in patientsof CPB, or hip replacement.

SUMMARY OF THE INVENTION

This invention is based on the discovery of peptides that inhibit serineproteases. Serine proteases such as, for example, kallikrein, areinvolved in, for example, pathways leading to excessive perioperativeblood loss and the onset of systemic inflammatory response. Preferredkallikrein peptide inhibitors include those described in U.S. Pat. Nos.6,333,402 and 6,057,287 to Markland et al., the contents of which areincorporated herein by reference in their entirety. The invention isdirected in part to the use of the peptides in therapeutic methods andcompositions suitable for use in eliminating or reducing variousischemias, including but not limited to perioperative blood loss, andthe onset of systemic inflammatory response. Perioperative blood lossresults from invasive surgical procedures that lead to contactactivation of complement components and the coagulation/fibrinolysissystems. More specifically, the invention provides methods of usingkallikrein inhibitors to reduce or prevent perioperative blood loss anda systemic inflammatory response in patients subjected to invasivesurgical procedures, especially cardiothoracic surgeries.

In one embodiment, the invention is directed to a method for preventingor reducing ischemia in a patient comprising administering to thepatient a composition comprising a polypeptide comprising the amino acidsequence: Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 GlyXaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly GlyCys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), whereinXaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are each individually anamino acid or absent; Xaa10 is an amino acid selected from the groupconsisting of: Asp and Glu; Xaa11 is an amino acid selected from thegroup consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 isan amino acid selected from the group consisting of: Arg, His, Pro, Asn,Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acid selected fromthe group consisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln;Xaa16 is an amino acid selected from the group consisting of: Ala, Gly,Ser, Asp and Asn; Xaa17 is an amino acid selected from the groupconsisting of: Ala, Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 is anamino acid selected from the group consisting of: His, Leu, Gln and Ala;Xaa19 is an amino acid selected from the group consisting of: Pro, Gln,Leu, Asn and Ile; Xaa21 is an amino acid selected from the groupconsisting of: Trp, Phe, Tyr, His and Ile; Xaa22 is an amino acidselected from the group consisting of: Tyr and Phe; Xaa23 is an aminoacid selected from the group consisting of: Tyr and Phe; Xaa31 is anamino acid selected from the group consisting of: Glu, Asp, Gln, Asn,Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected fromthe group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Glyand Val; Xaa34 is an amino acid selected from the group consisting of:Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is an amino acidselected from the group consisting of: Tyr, Trp and Phe; Xaa39 is anamino acid selected from the group consisting of: Glu, Gly, Ala, Ser andAsp; Xaa40 is an amino acid selected from the group consisting of: Glyand Ala; Xaa43 is an amino acid selected from the group consisting of:Asn and Gly; Xaa45 is an amino acid selected from the group consistingof: Phe and Tyr; and wherein the polypeptide inhibits kallikrein.

In a particular embodiment, the ischemia is perioperative blood loss dueto a surgical procedure performed on the patient. The surgical procedurecan be a cardiothoracic surgery, such as, for example, cardiopulmonarybypass or coronary artery bypass grafting.

In a particular embodiment, individual amino acid positions of SEQ IDNO:1 can be one or more of the following: Xaa10 is Asp, Xaa11 is Asp,Xaa13 is Pro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His,Xaa19 is Pro, Xaa21 is Trp, Xaa31 is Glu, Xaa32 is Glu, Xaa34 is Ile,Xaa35 is Tyr, Xaa39 is Glu.

In another embodiment, the invention is directed to a method forpreventing or reducing the onset of systemic inflammatory responseassociated with a surgical procedure in a patient comprisingadministering to the patient a composition comprising a polypeptidecomprising the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57Xaa58 (SEQ ID NO:1), wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 orXaa58 are each individually an amino acid or absent; Xaa10 is an aminoacid selected from the group consisting of: Asp and Glu; Xaa11 is anamino acid selected from the group consisting of: Asp, Gly, Ser, Val,Asn, Ile, Ala and Thr; Xaa13 is an amino acid selected from the groupconsisting of: Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln;Xaa15 is an amino acid selected from the group consisting of: Arg, Lys,Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is an amino acid selected fromthe group consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an aminoacid selected from the group consisting of: Ala, Asn, Ser, Ile, Gly,Val, Gln and Thr; Xaa18 is an amino acid selected from the groupconsisting of: His, Leu, Gln and Ala; Xaa19 is an amino acid selectedfrom the group consisting of: Pro, Gln, Leu, Asn and Ile; Xaa21 is anamino acid selected from the group consisting of: Trp, Phe, Tyr, His andIle; Xaa22 is an amino acid selected from the group consisting of: Tyrand Phe; Xaa23 is an amino acid selected from the group consisting of:Tyr and Phe; Xaa31 is an amino acid selected from the group consistingof: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is anamino acid selected from the group consisting of: Glu, Gln, Asp Asn,Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an amino acid selectedfrom the group consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu;Xaa35 is an amino acid selected from the group consisting of: Tyr, Trpand Phe; Xaa39 is an amino acid selected from the group consisting of:Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid selected from thegroup consisting of: Gly and Ala; Xaa43 is an amino acid selected fromthe group consisting of: Asn and Gly; Xaa45 is an amino acid selectedfrom the group consisting of: Phe and Tyr; and wherein the polypeptideinhibits kallikrein. In a particular embodiment, the surgical procedurecan be a cardiothoracic surgery, such as, for example, cardiopulmonarybypass or coronary artery bypass grafting. In a particular embodiment,individual amino acid positions of SEQ ID NO: 1 can be one or more ofthe following: Xaa10 is Asp, Xaa11 is Asp, Xaa13 is Pro, Xaa15 is Arg,Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His, Xaa19 is Pro, Xaa21 is Trp,Xaa31 is Glu, Xaa32 is Glu, Xaa34 is Ile, Xaa35 is Tyr, Xaa39 is Glu.

In yet another embodiment, the invention is directed to a method forpreventing or reducing the onset of systemic inflammatory responseassociated with a surgical procedure in a patient comprisingadministering to the patient a composition comprising a polypeptideconsisting of the amino acid sequence: Glu Ala Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ IDNO:2), wherein the polypeptide inhibits kallikrein. In one embodiment,the surgical procedure is a cardiothoracic surgery, such as, forexample, cardiopulmonary bypass or coronary artery bypass grafting.

In another embodiment, the invention is directed to a method forpreventing or reducing ischemia in a patient comprising administering tothe patient a composition comprising a polypeptide consisting of theamino acid sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp AspGly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg GlnCys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu SerLeu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2), wherein thepolypeptide inhibits kallikrein. In a particular embodiment, theischemia can be perioperative blood loss due to a surgical procedureperformed on the patient. In one embodiment, the surgical procedure is acardiothoracic surgery, such as, for example, cardiopulmonary bypass orcoronary artery bypass grafting.

In yet another embodiment, the invention is directed to a method forpreventing or reducing the onset of systemic inflammatory responseassociated with a surgical procedure in a patient comprisingadministering to the patient a composition comprising a polypeptideconsisting of the amino acid sequence: Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (amino acids3-60 of SEQ ID NO:2), wherein the polypeptide inhibits kallikrein. Inone embodiment, the surgical procedure is a cardiothoracic surgery, suchas, for example, cardiopulmonary bypass or coronary artery bypassgrafting.

In another embodiment, the invention is directed to a method forpreventing or reducing ischemia in a patient comprising administering tothe patient a composition comprising a polypeptide consisting of theamino acid sequence: Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly ProCys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu GluGlu Cys Lys Lys Met Cys Thr Arg Asp (amino acids 3-60 of SEQ ID NO:2),wherein the polypeptide inhibits kallikrein. In a particular embodiment,the ischemia can be perioperative blood loss due to a surgical procedureperformed on the patient. In one embodiment, the surgical procedure is acardiothoracic surgery, such as, for example, cardiopulmonary bypass orcoronary artery bypass grafting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified diagram of major multiple pathways and relatedevents involved in the contact activation system and systemicinflammatory response (SIR) that can arise in a patient subjected tosoft and bone tissue trauma such as that associated with a coronaryartery bypass grafting (CABG) procedure, especially when the CABGprocedure involves extra-corporeal blood circulation, such ascardiopulmonary bypass (Bypass Apparatus). Arrows indicate activationfrom one component or event to another component or event in thecascade. Arrows in both directions indicate activating effects ofcomponents or events in both directions. Broken arrows indicate likelyparticipation of one component or event in the activation of anothercomponent or event. Abbreviations are as follows: “tPA”−=tissueplasminogen activator; “C5a”=a protein component of the complementsystem; “fXIIa”=activator protein of prekallikrein to form activekallikrein; “Extrinsic”=extrinsic coagulation system;“Intrinsic”=intrinsic coagulation system.

FIG. 2 shows a portion of a DNA and corresponding deduced amino acid fora KI polypeptide of the invention in plasmid pPIC-K503. The inserted DNAencodes the mat.alpha. prepro signal peptide of Saccharomyces cerevisiae(underlined) fused in frame to the amino terminus of the PEP-1 KIpolypeptide having the amino acid sequence enclosed by the boxed area.The amino acid sequence of the PEP-1 KI polypeptide shown in the boxedregion is SEQ ID NO:2, and the corresponding nucleotide coding sequenceof the KI polypeptide is SEQ ID NO:3. The dashed arrows indicate thelocation and direction of two PCR primer sequences in AOX regions thatwere used to produce sequencing templates. DNA sequence for the entirenucleotide sequence of the figure comprises the structural codingsequence for the fusion protein and is designated SEQ ID NO:27. Theentire amino acid sequence is SEQ ID NO:28. The double underlinedportion of the sequence indicates a diagnostic probe sequence. BstBI andEcoRI indicate locations of their respective palindromic, hexameric,restriction endonuclease sites in the sequence. Asterisks denotetranslational stop codons.

FIGS. 3A and 3B show an alignment of amino acid sequences of thepreferred embodiments of the invention, the native LACI sequence fromwhich these variants were derived (SEQ ID NO:32), and other known Kunitzdomains (SEQ ID NOS:29-31 and 33-53). Cysteine residues are highlighted.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The invention is based on the discovery of a group of kallikreininhibitor (KI) polypeptides that inhibit plasma kallikrein with aspecificity that permits their use in improved methods of preventing orreducing ischemia such as, for example, perioperative blood loss and/ora systemic inflammatory response (SIR) induced by kallikrein,especially, for example, in patients undergoing surgical procedures andparticularly surgical procedures involving cardiothoracic surgery, e.g.,cardiopulmonary bypass (CPB), such as a coronary artery bypass graft(CABG) procedures. K's can be used specifically for, e.g., pediatriccardiac surgery, lung transplantation, total hip replacement andorthotopic liver transplantation, and to reduce or prevent perioperativestroke during CABG, extracorporeal membrane oxygenation (ECMO) andcerebrovascular accidents (CVA) during these procedures.

Cardiothoracic surgery is surgery of the chest area, most commonly theheart and lungs. Typical diseases treated by cardiothoracic surgeryinclude coronary artery disease, tumors and cancers of the lung,esophagus and chest wall, heart vessel and valve abnormalities, andbirth defects involving the chest or heart. Where cardiothoracic surgeryis utilized for treatment, the risk of blood loss (e.g., surgery-inducedischemia) and the onset of a systemic inflammatory response (SIR) isincurred. Surgery-induced SIR can result in severe organ dysfunction(systemic inflammatory response syndrome; SIRS).

Polypeptides Useful in the Invention

KI polypeptides useful in the invention comprise Kunitz domainpolypeptides. In one embodiment these Kunitz domains are variant formsof the looped structure comprising Kunitz domain 1 of humanlipoprotein-associated coagulation inhibitor (LACI) protein. LACIcontains three internal, well-defined, peptide loop structures that areparadigm Kunitz domains (Girard, T. et al., 1989. Nature, 338:518-520).The three Kunitz domains of LACI confer the ability to bind and inhibitkallikrein, although not with exceptional affinity. Variants of Kunitzdomain 1 of LACI described herein have been screened, isolated and bindkallikrein with enhanced affinity and specificity (see, for example,U.S. Pat. Nos. 5,795,865 and 6,057,287, incorporated herein byreference). An example of a preferred polypeptide useful in theinvention has the amino acid sequence defined by amino acids 3-60 of SEQID NO:2.

Every polypeptide useful in the invention binds kallikrein, andpreferred polypeptides are also kallikrein inhibitors (KI) as determinedusing kallikrein binding and inhibition assays known in the art. Theenhanced affinity and specificity for kallikrein of the variant Kunitzdomain polypeptides described herein provides the basis for their use incardiothoracic surgery, e.g., CPB and especially CABG surgicalprocedures, to prevent or reduce perioperative blood loss and/or theonset of SIR in patients undergoing such procedures. The KI polypeptidesused in the invention have or comprise the amino acid sequence of avariant Kunitz domain polypeptide originally isolated by screening phagedisplay libraries for the ability to bind kallikrein.

KI polypeptides useful in the methods and compositions of the inventioncomprise a Kunitz domain polypeptide comprising the amino acid sequence:

      Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13Cys (SEQ ID NO: 1) Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 GlyGly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58

“Xaa” refers to a position in a peptide chain that can be any of anumber of different amino acids. For example, for the KI peptidesdescribed herein, Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser,Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr,Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn orGln; Xaa16 can be Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser,Ile, Gly, Val, Gln or Thr; Xaa18 can be His, Leu, Gln or Ala; Xaa19 canbe Pro, Gln, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile;Xaa31 can be Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa23can be Glu, Gln, Asp, Asn Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 canbe Ile, Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp orPhe; Xaa39 can be Glu, Gly, Ser or Asp. Amino acids Xaa6, Xaa7, Xaa8,Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42,Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53, and Xaa54 can beany

amino acid. Additionally, each of the first four and at last three aminoacids of SEQ ID NO: 1 can optionally be present or absent and can be anyamino acid, if present.

Peptides defined according to SEQ ID NO: 1 form a set of polypeptidesthat bind to kallikrein. For example, in a preferred embodiment of theinvention, a KI polypeptide useful in the methods and compositions ofthe invention has the following variable positions: Xaa11 can be Asp,Gly, Ser or Val; Xaa13 can be Pro, Arg, His or Asn; Xaa15 can be Arg orLys; Xaa16 can be Ala or Gly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18can be His, Leu or Gln; Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trpor Phe; Xaa31 is Glu; Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr orSer; Xaa35 is Tyr; and Xaa39 can be Glu, Gly or Ala.

A more specific embodiment of the claimed invention is defined by thefollowing amino acids at variable positions: Xaa10 is Asp; Xaa11 is Asp;Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can be Ala or Gly; Xaa17 isAla; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp; Xaa31 is Glu; Xaa32 isGlu; Xaa34 can be Ile or Ser; Xaa35 is Tyr; and Xaa39 is Gly.

Also encompassed within the scope of the invention are peptides thatcomprise portions of the polypeptides described herein. For example,polypeptides could comprise binding domains for specific kallikreinepitopes. Such fragments of the polypeptides described herein would alsobe encompassed.

KI polypeptides useful in the methods and compositions described hereincomprise a Kunitz domain. A subset of the sequences encompassed by SEQID NO: 1 are described by the following (where not indicated, “Xaa”refers to the same set of amino acids that are allowed for SEQ ID NO:1):

(SEQ ID NO: 54)       Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn IlePhe Thr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.(amino acids 3-60 of SEQ ID NO: 2)       Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 4)      Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys AlaAsn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 5)       Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 6)       Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys LysAla Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln PheThr Tyr Gly Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu CysLys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 7)       Met His Ser Phe CysAla Phe Lys Ala Asp Asp Gly His Cys Lys Ala Ser Leu Pro Arg Phe Phe PheAsn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Gly Gly AsnGln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 8)       Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly HisCys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu GluGlu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 9)       Met His SerPhe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Gly Ala His Leu Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys GluGly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr ArgAsp, (SEQ ID NO: 10)       Met His Ser Phe Cys Ala Phe Lys Ala Asp AspGly Arg Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg GlnCys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu SerLeu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 11)       MetHis Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Gly Ala His ProArg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met CysThr Arg Asp, (SEQ ID NO: 12)       Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe ThrArg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg PheGlu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 13)      Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg GlyAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 14)       Met His Ser Phe Cys AlaPhe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala Gln Pro Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Gln Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 15)       Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Ser CysArg Ala Ala His Leu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 16)       Met His Ser PheCys Ala Phe Lys Ala Glu Gly Gly Ser Cys Arg Ala Ala His Gln Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Gln Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 17)       Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 18)       Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Arg Gly Ala Leu Pro ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO: 19)       Met His Ser Phe Cys Ala Phe Lys Ala AspSer Gly Asn Cys Arg Gly Asn Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 20)      Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg GlyAsn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 21)       Met His Ser Phe Cys AlaPhe Lys Ala Asp Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 22)       Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg CysArg Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp.

FIGS. 3A and 3B provides an amino acid sequence alignment of thesesequences, the native LACI sequence from which these variants werederived (SEQ ID NO:32), and other known Kunitz domains (SEQ ID NOS:29-31 and 33-53).

The KI polypeptides useful in the methods and compositions describedherein can be made synthetically using any standard polypeptidesynthesis protocol and equipment. For example, the stepwise synthesis ofa KI polypeptide described herein can be carried out by the removal ofan amino (N) terminal-protecting group from an initial (i.e.,carboxy-terminal) amino acid, and coupling thereto of the carboxyl endof the next amino acid in the sequence of the polypeptide. This aminoacid is also suitably protected. The carboxyl group of the incomingamino acid can be activated to react with the N-terminus of the boundamino acid by formation into a reactive group such as formation into acarbodiimide, a symmetric acid anhydride, or an “active ester” groupsuch as hydroxybenzotriazole or pentafluorophenyl esters. Preferredsolid-phase peptide synthesis methods include the BOC method, whichutilizes tert-butyloxycarbonyl as the .alpha.-amino protecting group,and the FMOC method, which utilizes 9-fluorenylmethloxycarbonyl toprotect the .alpha.-amino of the amino acid residues. Both methods arewell known to those of skill in the art (Stewart, J. and Young, J.,Solid-Phase Peptide Synthesis (W. H. Freeman Co., San Francisco 1989);Merrifield, J., 1963. Am. Chem. Soc., 85:2149-2154; Bodanszky, M. andBodanszky, A., The Practice of Peptide Synthesis (Springer-Verlag, NewYork 1984), the entire teachings of these references is incorporatedherein by reference). If desired, additional amino- and/orcarboxy-terminal amino acids can be designed into the amino acidsequence and added during polypeptide synthesis.

Alternatively, Kunitz domain polypeptides and KI polypeptides useful inthe compositions and methods of the invention can be produced byrecombinant methods using any of a number of cells and correspondingexpression vectors, including but not limited to bacterial expressionvectors, yeast expression vectors, baculovirus expression vectors,mammalian viral expression vectors, and the like. Kunitz domainpolypeptides and KI polypeptides useful in the compositions and methodsof the invention can also be produced transgenically using nucleic acidmolecules comprising a coding sequence for a Kunitz domain or KIpolypeptide described herein, wherein the nucleic acid molecule can beintegrated into and expressed from the genome of a host animal usingtransgenic methods available in the art. In some cases, it could benecessary or advantageous to fuse the coding sequence for a Kunitzdomain polypeptide or a KI polypeptide comprising the Kunitz domain toanother coding sequence in an expression vector to form a fusionpolypeptide that is readily expressed in a host cell. Preferably, thehost cell that expresses such a fusion polypeptide also processes thefusion polypeptide to yield a Kunitz domain or KI polypeptide useful inthe invention that contains only the desired amino acid sequence.Obviously, if any other amino acid(s) remain attached to the expressedKunitz domain or KI polypeptide, such additional amino acid(s) shouldnot diminish the kallikrein binding and/or kallikrein inhibitoryactivity of the Kunitz domain or KI polypeptide so as to preclude use ofthe polypeptide in the methods or compositions of the invention.

A preferred recombinant expression system for producing KI polypeptidesuseful in the methods and compositions described herein is a yeastexpression vector, which permits a nucleic acid sequence encoding theamino acid sequence for a KI polypeptide or Kunitz domain polypeptide tobe linked in the same reading frame with a nucleotide sequence encodingthe mat.alpha. prepro leader peptide sequence of Saccharomycescerevisiae, which in turn is under the control of an operable yeastpromoter. The resulting recombinant yeast expression plasmid can then betransformed by standard methods into the cells of an appropriate,compatible yeast host, which cells are able to express the recombinantprotein from the recombinant yeast expression vector. Preferably, a hostyeast cell transformed with such a recombinant expression vector is alsoable to process the fusion protein to provide an active KI polypeptideuseful in the methods and compositions of the invention. A preferredyeast host for producing recombinant Kunitz domain polypeptides and KIpolypeptides comprising such Kunitz domains is Pichia pastoris.

As noted above, KI polypeptides that are useful in the methods andcompositions described herein can comprise a Kunitz domain polypeptidedescribed herein. Some KI polypeptides can comprise an additionalflanking sequence, preferably of one to six amino acids in length, atthe amino and/or carboxy-terminal end, provided such additional aminoacids do not significantly diminish kallikrein binding affinity orkallikrein inhibition activity so as to preclude use in the methods andcompositions described herein. Such additional amino acids can bedeliberately added to express a KI polypeptide in a particularrecombinant host cell or can be added to provide an additional function,e.g., to provide a peptide to link the KI polypeptide to anothermolecule or to provide an affinity moiety that facilitates purificationof the polypeptide. Preferably, the additional amino acid(s) do notinclude cysteine, which could interfere with the disulfide bonds of theKunitz domain.

An example of a preferred Kunitz domain polypeptide useful in themethods and compositions of the invention has the amino acid sequence ofresidues 3-60 of SEQ ID NO:2. When expressed and processed in a yeastfusion protein expression system (e.g., based on the integratingexpression plasmid pHIL-D2), such a Kunitz domain polypeptide retains anadditional amino terminal Glu-Ala dipeptide from the fusion with themat.alpha. prepro leader peptide sequence of S. cerevisiae. Whensecreted from the yeast host cell, most of the leader peptide isprocessed from the fusion protein to yield a functional KI polypeptide(referred to herein as “PEP-1”) having the amino acid sequence of SEQ IDNO:2 (see boxed region in FIG. 2).

Particularly preferred KI polypeptides useful in the methods andcompositions described herein have a binding affinity for kallikreinthat is on the order of 1000 times higher than that of aprotinin, whichis currently approved for use in CABG procedures to reduce blood loss.The surprisingly high binding affinities of such KI polypeptidesdescribed herein indicate that such KI polypeptides exhibit a highdegree of specificity for kallikrein to the exclusion of other moleculartargets (see Table 1, below). Thus, use of such polypeptides accordingto the invention reduces much of the speculation as to the possibletherapeutic targets in a patient. The lower degree of specificityexhibited by, for example, aprotinin, leads to possible pleiotropic sideeffects and ambiguity as to its therapeutic mechanism.

The polypeptides defined by, for example, SEQ ID NO: 1 contain invariantpositions, e.g., positions 5, 14, 30, 51 and 55 can be Cys only. Otherpositions such as, for example, positions 6, 7, 8, 9, 20, 24, 25, 26,27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53 and 54 can be anyamino acid (including non-naturally occurring amino acids). In aparticularly preferred embodiment, one or more amino acids correspond tothat of a native sequence (e.g., SEQ ID NO:32, see FIG. 3). In apreferred embodiment, at least one variable position is different fromthat of the native sequence. In yet another preferred embodiment, theamino acids can each be individually or collectively substituted by aconservative or non-conservative amino acid substitution. Conservativeamino acid substitutions replace an amino acid with another amino acidof similar chemical structure and may have no affect on proteinfunction. Non-conservative amino acid substitutions replace an aminoacid with another amino acid of dissimilar chemical structure. Examplesof conserved amino acid substitutions include, for example, Asn->Asp,Arg->Lys and Ser->Thr. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 of theseamino acids can be independently or collectively, in any combination,selected to correspond to the corresponding position of SEQ ID NO:2.

Other positions, for example, positions 10, 11, 13, 15, 16, 17, 18, 19,21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, can be any of a selectedset of amino acids. Thus SEQ ID NO: 1 defines a set of possiblesequences. Each member of this set contains, for example, a cysteine atpositions 5, 14, 30, 51 and 55, and any one of a specific set of aminoacids at positions 10, 11, 13, 15, 16, 17, 18, 19, 221, 22, 23, 31, 32,34, 35, 39, 40, 43 and 45. In a preferred embodiment, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and/or 19 of these aminoacids can be independently or collectively, in any combination, selectedto correspond to the corresponding position of SEQ ID NO:2. The peptidepreferably has at least 80%, at least 85%, at least 90% or at least 95%identity to SEQ ID NO:2.

Methods and Compositions

The present invention is also directed to methods for preventing orreducing ischemia. Preferred in the invention are methods for preventingor reducing perioperative blood loss and/or a systemic inflammatoryresponse (SIR) in a patient, especially associated with cardiothoracicsurgery. A method for treatment involves the administration of a KIpolypeptide comprising a Kunitz domain. One embodiment of the methodinvolves using a peptide containing an amino acid sequence of SEQ ID NO:1 that has an affinity for kallikrein that is approximately 1000-fold ormore higher than that of a broad range serine protease, e.g., aprotinin,which is isolated from bovine lung and currently approved for use inCABG procedures (TRASYLOL.RTM., Bayer Corporation PharmaceuticalDivision, West Haven, Conn.).

Patients subjected to any of a number of surgical procedures, especiallythose involving extra-corporeal circulation, e.g., cardiothoracicsurgery, such as, for example, CPB, and/or bone trauma, such as sternalsplit or hip replacement, are at risk for perioperative blood loss andinflammation. Contact of a patient's blood with the cut surfaces of boneor of CPB equipment is sufficient to activate one or several undesirablecascade responses, including a contact activation system (CAS), whichcan lead to extensive perioperative blood loss requiring immediate bloodtransfusion, as well as a systemic inflammatory response (SIR), which,in turn, can result in permanent damage to tissues and organs. While notdesiring to be limited to any particular mechanism or theory, it appearsthat the blood loss that occurs associated with cardiothoracic surgery,e.g., CPB, as in a CABG procedure, probably results from extensivecapillary leakage, which can result in significant loss of blood thatmust be replaced by immediate blood transfusion.

The methods described herein are useful for preventing or reducingvarious ischemias including, for example, perioperative blood loss andSIR in a patient subjected to a surgical procedure, and especiallywherein the surgical procedure requires extra-corporeal circulation,e.g., cardiothoracic surgery, such as, for example, CPB. The methods ofthe invention are particularly useful for preventing or reducingperioperative blood loss and/or SIR in a patient subjected to a CABGprocedure requiring CPB or other cardiac surgery.

Preferred compositions for medical use comprise a KI polypeptidedescribed herein. Such compositions useful can further comprise one ormore pharmaceutically acceptable buffers, carriers, and excipients,which can provide a desirable feature to the composition including, butnot limited to, enhanced administration of the composition to a patient,enhanced circulating half-life of the KI polypeptide of the composition,enhanced compatibility of the composition with patient blood chemistry,enhanced storage of the composition, and/or enhanced efficacy of thecomposition upon administration to a patient. In addition to a KIpolypeptide described herein, compositions can further comprise one ormore other pharmaceutically active compounds that provide an additionalprophylactic or therapeutic benefit to a patient of an invasive surgicalprocedure.

Compositions useful in the methods of the invention comprise any of theKunitz domain polypeptides or KI polypeptides comprising such Kunitzdomain polypeptides described herein. Particularly preferred are KIpolypeptides comprising a Kunitz domain polypeptide having a 58-aminoacid sequence of amino acids 3-60 of SEQ ID NO:2. An example of such aparticularly preferred KI polypeptide useful in the methods andcompositions of the invention is the PEP-1 KI polypeptide having the60-amino acid sequence of SEQ ID NO:2. A nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:2 is provided in SEQ ID NO:3 (see,e.g., nucleotides 309-488 in FIG. 2). It is understood that based on theknown genetic code, the invention also provides degenerate forms of thenucleotide sequence of SEQ ID NO:3 by simply substituting one or more ofthe known degenerate codons for each amino acid encoded by thenucleotide sequence. Nucleotides 7-180 of SEQ ID NO:3, and degenerateforms thereof, encode the non-naturally occurring Kunitz domainpolypeptide having the 58-amino acid sequence of amino acids 3-60 of SEQID NO:2.

Any of a variety of nucleic acid molecules can comprise the nucleotidesequence of nucleotides 7-180 of SEQ ID NO:3, degenerate forms, andportions thereof, including but not limited to, recombinant phagegenomes, recombinant mammalian viral vectors, recombinant insect viralvectors, yeast mini chromosomes, and various plasmids. Such plasmidsinclude those used to clone and/or express such nucleotide codingsequences. Expression vectors provide a promoter, which can be operablylinked to a particular nucleotide sequence and an appropriate host cell,which is able to transcribe the particular nucleotide coding sequenceinto a functional messenger RNA (mRNA) and also translate the mRNA intothe corresponding polypeptide. A polypeptide so produced can then beisolated from the host cell. Nucleic acid molecules comprising a nucleicacid sequence encoding a Kunitz domain or KI polypeptide describedherein can be made by standard nucleic acid synthesis methods,recombinant DNA methodologies, polymerase chain reaction (PCR) methods,and any combination thereof.

Perioperative Blood Loss and Reduced Heart Bloodflow

Due to the many advances in medicine, a number of highly invasivesurgical procedures are carried out each day that result in blood loss,or place patients at a high risk for blood loss. Such patients must becarefully monitored to restore and maintain normal blood supply andhemostasis, and they may need blood transfusions. Surgical proceduresthat involve blood loss include those involving extra-corporealcirculation methods such as cardiothoracic surgery, e.g., CPB. In suchmethods, a patient's heart is stopped and the circulation, oxygenation,and maintenance of blood volume are carried out artificially using anextra-corporeal circuit and a synthetic membrane oxygenator. Thesetechniques are commonly used during cardiac surgery. Additionally, it isapparent that surgery involving extensive trauma to bone, such as thesternal split necessary in CABG or hip replacement procedures, is alsoassociated with activation of the CAS, which can result in a variety ofdisruptions in the blood and vasculature.

Atherosclerotic coronary artery disease (CAD) causes a narrowing of thelumen of one or several of the coronary arteries; this limits the flowof blood to the myocardium (i.e., the heart muscle) and can causeangina, heart failure, and myocardial infarcts. In the end stage ofcoronary artery atherosclerosis, the coronary circulation can be almostcompletely occluded, causing life threatening angina or heart failure,with a very high mortality. CABG procedures may be required to bridgethe occluded blood vessel and restore blood to the heart; these arepotentially life saving. CABG procedures are among the most invasive ofsurgeries in which one or more healthy veins or arteries are implantedto provide a “bypass” around the occluded area of the diseased vessel.CABG procedures carry with them a small but important perioperativerisk, but they are very successful in providing patients with immediaterelief from the mortality and morbidity of atheroscleroticcardiovascular disease. Despite these very encouraging results, repeatCABG procedures are frequently necessary, as indicated by a clearincrease in the number of patients who eventually undergo second andeven third procedures; the perioperative mortality and morbidity seen inprimary CABG procedures is increased in these re-do procedures.

There have been improvements in minimally invasive surgical techniquesfor uncomplicated CAD. However, nearly all CABG procedures performed forvalvular and/or congenital heart disease, heart transplantation, andmajor aortic procedures, are still carried out on patients supported byCPB. In CPB, large cannulae are inserted into the great vessels of apatient to permit mechanical pumping and oxygenation of the blood usinga membrane oxygenator. The blood is returned to the patient withoutflowing through the lungs, which are hypoperfused during this procedure.The heart is stopped using a cardioplegic solution, the patient cooledto help prevent brain damage, and the peripheral circulating volumeincreased by an extracorporeal circuit, i.e., the CPB circuit, whichrequires “priming” with donor blood and saline mixtures are used to fillthe extracorporeal circuit. CPB has been extensively used in a varietyof procedures performed for nearly half a century with successfuloutcomes. The interaction between artificial surfaces, blood cells,blood proteins, damaged vascular endothelium, and extravascular tissues,such as bone, disturbs hemostasis and frequently activates the CAS,which, as noted above, can result in a variety of disruptions in theblood and vasculature. Such disruption leads to excess perioperativebleeding, which then requires immediate blood transfusion. A consequenceof circulating whole blood through an extracorporeal circuit in CPB canalso include the systemic inflammatory response (SIR), which isinitiated by contact activation of the coagulation and complementsystems. Indeed, much of the morbidity and mortality associated withseemingly mechanically successful CPB surgical procedures is the resultof the effects of activating coagulation, fibrinolysis, or complementsystems. Such activation can damage the pulmonary system, leading toadult respiratory distress syndrome (ARDS), impairment of kidney andsplanchnic circulation, and induction of a general coagulopathy leadingto blood loss and the need for transfusions. In addition to the dangersof perioperative blood loss, additional pathologies associated with SIRinclude neurocognitive deficits, stroke, renal failure, acute myocardialinfarct, and cardiac tissue damage.

Blood transfusions also present a significant risk of infection andelevate the cost of CABG or other similar procedures that require CPB.In the absence of any pharmacological intervention, three to seven unitsof blood must typically be expended on a patient, even with excellentsurgical techniques. Accordingly, there is considerable incentive forthe development of new and improved pharmacologically effectivecompounds to reduce or prevent perioperative bleeding and SIR inpatients subjected to CPB and CABG procedures.

Administration and Dosing Considerations for KI Polypeptides

KI polypeptides described herein can be administered to a patientbefore, during, and/or after a surgical procedure in a pharmaceuticallyacceptable composition. The term “pharmaceutically acceptable”composition refers to a non-toxic carrier or excipient that may beadministered to a patient, together with a compound of this invention,and wherein the carrier or excipient not destroy the biological orpharmacological activity of the composition. KI polypeptides describedherein can be administered locally or systemically by any suitable meansfor delivery of a kallikrein inhibitory amount of the KI polypeptides toa patient including but not limited to systemic administrations such as,for example, intravenous and inhalation. Parenteral administration isparticularly preferred.

For parenteral administration, the polypeptides can be injectedintravenously, intramuscularly, intraperitoneally, or subcutaneously.Intravenous administration is preferred. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Other pharmaceutically acceptable carriers include, but are notlimited to, sterile water, saline solution, and buffered saline(including buffers like phosphate or acetate), alcohol, vegetable oils,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, paraffin, etc. Where necessary, the composition canalso include a solubilizing agent and a local anaesthetic such aslidocaine to ease pain at the site of the injection, preservatives,stabilizers, wetting agents, emulsifiers, salts, lubricants, etc. aslong as they do not react deleteriously with the active compounds.Similarly, the composition can comprise conventional excipients, e.g.,pharmaceutically acceptable organic or inorganic carrier substancessuitable for parenteral, enteral or intranasal application which do notdeleteriously react with the active compounds. Generally, theingredients will be supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent in activity units.Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade “water for injection” or saline. Where the composition is to beadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

Preferably, the methods of the invention comprise administering a KIpolypeptide to a patient as an intravenous infusion according to anyapproved procedure. Thus, a KI polypeptide described herein can beadministered to a patient subjected to a CABG procedure at the timessimilar to those currently used in approved protocols for administeringaprotinin and in an amount necessary to provide a patient with arequired number or concentration of kallikrein inhibitory units (KIU).According to the invention, a KI polypeptide described herein can alsobe administered to a patient in the immediate postoperative period, whenbleeding abnormalities can occur as a consequence of downstream effectsof SIR. For example, in a procedure involving CPB, a KI polypeptidedescribed herein can be administered to a patient as an initial loadingdose, e.g., an effective amount over the course of a convenient time,such as 10 minutes, prior to induction of anesthesia. Then, at inductionof anesthesia, a second dose of KI polypeptide can be injected into theCPB priming fluid (“pump prime volume”). The patient can then be placedon a continuous and controlled intravenous infusion dose for theduration of the surgical procedure, and after the procedure ifindicated.

Currently there are two regimens approved in the United States foradministering aprotinin to a patient undergoing a CABG procedure (see,product label and insert for TRASYLOL.RTM., Bayer CorporationPharmaceutical Division, West Haven, Conn.). One such approved regimenuses a 2 million KIU intravenous loading dose, 2 million KIU into thepump prime volume, and 500,000 KIU per hour of surgery. Another approvedregimen uses 1 million KIU intravenous loading dose, 1 million KIU intothe pump prime volume, and 250,000 KIU per hour of surgery. As theseregimens are based on KIU, the regimens are readily adapted to any KIpolypeptide described herein once the specific activity and KIU of aparticular KI polypeptide has been determined by standard assays. Owingto the enhanced binding affinity and inhibitory activity inrepresentative KI polypeptides described herein relative to aprotinin,it is expected that such compositions and methods of the invention arelikely to require fewer milligrams (mg) per patient to provide a patientwith the required number or concentration of KIU.

Several considerations regarding dosing with a KI polypeptide in methodsof the invention can be illustrated by way of example with therepresentative PEP-1 KI polypeptide of the invention having the aminosequence of SEQ ID NO:2 (molecular weight of 7,054 Daltons).

Table 1, below, provides a comparison of the affinity (K.sub.i,app) ofthe PEP-1 KI polypeptide for kallikrein and eleven other known plasmaproteases.

1TABLE 1 Aprotinin Protease Substrate PEP-1 K.sub.i,app (pM) K.sub.i,app(pM) human plasma kallikrein 44 3.0 times. 10.sup.4 human urinekallikrein>1 .times. 10.sup.8 4.0 .times. 10.sup.3 porcine pancreatickallikrein 2.7 .times. 10.sup.7 550 human C1r, activated>2.0 .times.10.sup.8>1.0 .times. 10.sup.7 human C1s, activated>2.0 .times.10.sup.7>1.0 .times. 10.sup.8 human plasma factor XIa 1.0 .times.10.sup.4 ND human plasma factor XIIa>2.0 .times. 10.sup.7>1.0 .times.10.sup.8 human plasmin 1.4 .times. 10.sup.5 894 human pancreatictrypsin>2 .times. 10.sup.7 ND human pancreatic chymotrypsin>2.0 .times.10.sup.7 7.3 .times. 10.sup.5 human neutrophil elastase>2.0 .times.10.sup.7 1.7 .times. 10.sup.6 human plasma thrombin>2.0 .times.10.sup.7 >1.0 .times. 10.sup.8 ND=not determined

Clearly, the PEP-1 KI polypeptide is highly specific for human plasmakallikrein. Furthermore, the affinity (K.sub.i,app) of PEP-1 forkallikrein is 1000 times higher than the affinity of aprotinin forkallikrein: the K.sub.i,app of PEP-1 for kallikrein is about 44 pM(Table 1), whereas the K.sub.i,app of aprotinin for kallikrein is 30,000pM. Thus, a dose of PEP-1 could be approximately 1000 times lower thanthat used for aprotinin on a per mole basis. However, consideration ofseveral other factors may provide a more accurate estimation of the doseof PEP-1 required in practice. Such factors include the amount ofkallikrein activated during CPB in a particular patient, theconcentration of kallikrein required to elicit an SIR, and thebioavailability and pharmacological distribution of PEP-1 in a patient.Nevertheless, use of a KI polypeptide in methods according to theinvention and provided in doses currently approved for the use ofaprotinin is still expected to provide significant improvements over thecurrent use of the less specific, lower affinity, bovine aprotinin.

For example, the total amount of circulating prekallikrein in plasma isestimated at approximately 500 nM (Silverberg, M. et al., “The ContactSystem and Its Disorders,” in Blood: Principles and Practice ofHematology, Handin, R. et al., eds., J B Lippincott Co., Philadelphia,1995). If all of the prekallikrein were activated, then at least 500 nMof PEP-1 would be required for a stoichiometric inhibition ofkallikrein. An individual having 5 liters of plasma would thereforerequire about 18 mg of PEP-1 to achieve a plasma concentration of 500nM.

Another factor to consider is the threshold concentration of kallikreinrequired to induce a SIR in a patient. If the concentration of activekallikrein must be maintained below, e.g., 1 nM, then owing to its highaffinity for kallikrein, PEP-1 offers a significant advantage overaprotinin in the amount of protein that would be required to inhibitSIR. In particular, a concentration of PEP-1 of 1 nM would inhibit 99.6%of kallikrein present at 1 nM (i.e., only 0.4 pM free kallikreinremaining in the blood), whereas, an aprotinin concentration of 1 nMwould only inhibit 24.5% of the kallikrein present at 1 nM. Foraprotinin to inhibit 99% of the kallikrein at 1 nM, an aprotininconcentration in the plasma of at least 3.mu.M is required (i.e., 3000times higher concentration than for PEP-1).

For a patient undergoing CPB, an initial clinical dose of PEP-1 can beestimated from a recommended dose regimen of aprotinin (1.times.10.sup.6KIU) mentioned above. Aprotinin is reported in a package insert to haveas specific inhibitory activity of 7143 KIU/mg determined using a dogblood pressure assay. Therefore, 1.times.10.sup.6 KIU of aprotinin isequivalent to 140 mg of aprotinin (i.e., 1.times.10.sup.6 KIU/7143KIU/mg=140 mg of aprotinin). In a patient having a blood plasma volumeof 5 liters, 140 mg corresponds to approximately 4.3.mu.M aprotinin(molecular weight of aprotinin is 6512 Daltons). The specific activityof aprotinin in the standard inhibitory assay used for PEP-1 is 0.4KIU/mg of polypeptide. A dose of 140 mg would correspond to a loadingdose for aprotinin of 56 KIU (140 mg.times.0.4 KIU/mg=56 KIU). Incontrast, since the specific activity of the PEP-1 KI polypeptide is 10KIU/mg in the standard inhibition assay, a dose of only 5.6 mg of PEP-1would be required to provide the number of KIUs equivalent to 140 mg ofaprotinin. In a patient with a plasma volume of 5 liters, thiscorresponds to about 160 nM PEP-1 (molecular weight of PEP-1 is 7054Daltons), although a higher dose of the PEP-1 KI polypeptide can berequired if all of the plasma kallikrein (500 nM) is activated and/or ifthis KI polypeptide is poorly distributed in a patient.

Furthermore, the KI polypeptides can be non-naturally occurring, andthey can be produced synthetically or recombinantly, as noted above,thereby avoiding potential contamination of transmissible diseases thatcan arise during isolation of a protein from a natural animal source,such as in the case of aprotinin, which is isolated from bovine lung.Increasingly important to administrative and public acceptance of atreatment or pharmaceutical composition comprising a polypeptide is theavoidance of possible contamination with and transmission to humanpatients of various pathological agents. Of particular interest for thesafety of proteins isolated from a bovine tissue is the elimination ofthe possible risk of exposure to viral mediated diseases, bacterialmediated diseases, and, especially, transmissible bovine spongiformencephalopathies.

As variants of the Kunitz domain 1 of the human LACI protein, fewer sideeffects are expected from administering the KI polypeptides to patientsthan for aprotinin, which is a bovine protein that is documented tocause anaphylactic and anaphylactoid responses in patients, especiallyin repeat administrations, such as second time CABG procedures.Additionally, the highly specific binding of the KI polypeptidesdescribed herein to kallikrein will effectively limit or eliminate thethrombotic tendencies observed with aprotinin, and reduce the problemsobserved with graft patency following CABG procedures.

The invention will be further described with reference to the followingnon-limiting examples. The teachings of all the patents, patentapplications and all other publications and websites cited herein areincorporated by reference in their entirety.

EXEMPLIFICATION Example 1 A Representative KI Polypeptide

A non-naturally occurring, KI polypeptide useful in the compositions andmethods of the invention was identified as a kallikrein bindingpolypeptide displayed on a recombinant phage from a phage displaylibrary. PEP-1 has the following amino acid sequence: Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly CysGlu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp (SEQ ID NO:2). The molecular weight of PEP-1 is 7,054 Daltons.

The nucleotide sequence (SEQ ID NO:3) encoding the PEP-1 amino acidsequence (SEQ ID NO:2), was derived from a peptide that was isolated andsequenced by standard methods determined from the recombinant phage DNA.PEP-1 was produced in amounts useful for further characterization as arecombinant protein in His4.sup.-phenotype host cells of yeast strainPichia pastoris.

Example 2 Construction of a Recombinant Plasmid to Express KIPolypeptides

The initial plasmid, pHIL-D2, is ampicillin resistant and contains awild-type allele of His4 from P. pastoris. The final DNA sequencecomprising the coding sequence for the mat.alpha. Prepro-PEP-1 fusionprotein in the recombinant expression plasmid pPIC-K503 is shown in FIG.2. The DNA sequence of pHIL-D2 was modified to produce pPIC-K503, asfollows:

1. The BstBI site in the 3′ AOX1 region of pHIL-D2, located downstreamof the His4 gene, was removed by partial restriction digestion, fill-in,and ligation, altering the sequence from TTCGAA (SEQ ID NO:23) toTTCGCGAA (SEQ ID NO:24). This modification was made to facilitate anddirect the cloning of the expression cassette into the plasmid.

2. The AatII site bearing the bla gene located downstream of His4 wasremoved by restriction digestion, fill-in, and ligation modifying thesequence from GACGTC (SEQ ID NO:25) to GACGTACGTC (SEQ ID NO:26). Thismodification was made to facilitate the cloning of expression cassetteshaving AatII sites into the plasmid. The DNA encoding PEP-1 wassynthesized based on the nucleotide sequence from the originalkallikrein-binding display phage and consisted of 450 base pairs (bp).The final DNA sequence of the insert in the pHIL-D2 plasmid is flankedby a 5′ AOX1 sequence and a 3′ AOX1 sequence (portions of which areshown in FIG. 2) and encode a fusion protein comprising the mat.alpha.prepro signal peptide of S. cerevisiae fused to the structural codingsequence for the PEP-1 KI polypeptide. The signal peptide was added tofacilitate the secretion of PEP-1 from the yeast host cells. Theoligonucleotides to form the insert were synthesized and obtainedcommercially (Genesis Labs, The Woodlands, Tex.), and the insert wasgenerated by polymerase chain reaction (PCR). The linked synthetic DNAencoding the mat.alpha. prepro/PEP-1 fusion protein was thenincorporated by ligation into the modified pHIL-D2 plasmid between theBstBI and EcoRI sites.

The ligation products were used to transform Escherichia coli strain XL1Blue. A PCR assay was used to screen E. coli transformants for thedesired plasmid construct. DNA from cell extracts was amplified by PCRusing primers containing the 5′ AOX1 and 3′ AOX1 sequences (see aboveand FIG. 2). PCR products of the correct number of base pairs weresequenced. In addition, approximately 20-50 bp on either side of thecloning sites were sequenced, and the predicted sequence was obtained.The final DNA sequence of the insert in the pHIL-D2 plasmid (to yieldplasmid pPIC-K503) is shown in FIG. 2 along with portions of flanking 5′and 3′ AOX1 sequences and corresponding amino acid sequence of thefusion protein comprising the mat.alpha. prepro signal peptide of S.cerevisiae fused to the structural coding sequence for the PEP-1 KIpolypeptide. A transformant with the desired expression plasmidconstruct, plasmid pPIC-K503, was selected for preparing yeast celllines for routine production of PEP-1.

Example 3 Manufacture of PEP-1 from Recombinant Yeast Cell Line

Spheroplasts of P. pastoris GS115 having the His4.sup.-phenotype weretransformed with the expression plasmid pPIC-K503 (above) followinglinearization of the plasmid at the SacI site and homologousrecombination of the plasmid DNA into the host 5′ AOX1 locus. Thephenotype of the production strain is His4.sup.+. The entire plasmid wasinserted into the 5′ AOX1 genomic sequence of the yeast.

Isolates from the transformation were screened for growth in the absenceof exogenous histidine with methanol as the sole carbon source. Greaterthan 95% of the transformants retained the wild-type ability to growwith methanol as the sole carbon source, thereby demonstrating that theplasmid had been inserted into the host genome by homologousrecombination rather than transplacement. These transformants did notrequire exogenous histidine for growth, thereby demonstrating that theplasmid had integrated into the host genome. Selected colonies werecloned. Small culture expression studies were performed to identifyclones secreting the highest levels of active PEP-1 into the culturemedium. PEP-1 secretion levels in clarified culture supernatantsolutions were quantified for PEP-1 levels by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and evaluated forkallikrein inhibition. A yeast clone was selected for PEP-1 productionbased on its high level of PEP-1 expression among cultures sampled.

Master and working cell banks of P. pastoris producing PEP-1 wereprepared commercially (MDS Pharma Services, Bothell, Wash.). A standardproduction of PEP-1 in yeast comprised three steps as follows: (1)preparation of the seed culture, (2) fermentation, and (3) recovery ofthe culture.

The seed culture step consisted of the inoculation of six flasks (300 mLeach) containing sterile inoculum broth (yeast nitrogen base, potassiumphosphate, and glycerol, pH=5) with the contents of a single vial of aworking cell bank of P. pastoris producing PEP-1. Flasks were inoculatedin an orbital shaker (300 rpm) for approximately 13 hours at 30.degree.C..+−.2.degree. C.

Fermentations were performed in a closed 100 liter Braun fermenterfilled with sterile broth. Each fermentation was initiated with thetransfer of the contents of the six seed culture flasks to thefermenter. After approximately 24 hours, the glycerol in the fermenterbecame exhausted and additional glycerol was added for approximately 8additional hours.

A mixed feed phase, which lasted approximately 83 hours, was theninitiated by the addition of a glycerol and methanol feed. At the end ofthis time, the fermentation was terminated, and the fermenter contentswere diluted with purified water. The purification and processing ofPEP-1 consisted of five steps as follows: (1) expanded bedchromatography, (2) cation exchange chromatography, (3) hydrophobicinteraction chromatography (HIC), (4) ultrafiltration and diafiltration,and (5) final filtration and packaging.

The initial purification step consisted of expanded bed chromatography.The diluted fermenter culture was applied to the equilibrated columnpacked with Streamline SP resin (Amersham Pharmacia Streamline 200chromatography column, Amersham Pharmacia, Piscataway, N.J.). The columnwas then washed (50 mM acetic acid, pH=3.0-3.5) in an up-flow mode toflush the yeast cells from the expanded bed. The top adaptor was raisedabove the expanded bed enhance washing. The flow was stopped and the bedwas allowed to settle. The adaptor was moved down so that it wasslightly above the settled bed. The direction of the flow was reversed.The effluent was collected. Washing was continued in a downward modeusing 50 mM sodium acetate, pH 4.0. The effluent was collected. PEP-1was eluted from the column using 50 mM sodium acetate, pH 6.0. Theeluate was collected in a 50 liter container. The eluate was thenfiltered through a 0.22.mu. filter into a clean container located in thepurification site. Additional samples were collected for thedetermination of PEP-1 concentration. A cation exchange chromatographystep was then performed using the filtered eluate from the expanded bedcolumn. PEP-1 was eluted from the column using 15 mM trisodium citrate,pH 6.2.

Additional proteins were removed from the PEP-1 preparation byhydrophobic interaction chromatography (HIC). Prior to HIC, the eluatefrom the cation exchange column was diluted with ammonium sulfate. Theeluate was applied to the column, and the PEP-1 was eluted usingammonium sulfate (0.572 M) in potassium phosphate (100 mM), pH 7.0. Theeluate was collected in fractions based on A280 values. All fractionswere collected into sterile, pre-weighed PETG bottles.

Selected fractions were pooled into a clean container. The pool wasconcentrated by ultrafiltration. The concentrated PEP-1 preparation wasimmediately diafiltered against ten volumes of PBS, pH 7.0.

A final filtration step was performed prior to packaging in order tominimize the bioburden in the bulk PEP-1. The bulk solution was filteredthrough a 0.22.mu. filter and collected into a sterile, pre-weighed PETGbottle. A sample was removed for lot release testing. The remainder ofthe bulk was dispensed aseptically into sterile PETG bottles and storedat −20.degree. C.

Example 4 Kallikrein Inhibition Assay

A kinetic test was used to measure inhibitory activity of KIpolypeptides, such as PEP-1. The kinetic assay measures fluorescencefollowing kallikrein-mediated cleavage of a substrate,prolylphenylalanylarginyl amino methyl coumarin. A known amount ofkallikrein was incubated with a serially diluted KI polypeptidereference standard or serially diluted KI polypeptide test samples, in asuitable reaction buffer on a microtiter plate. Each sample was run intriplicate. The substrate solution was added, and the plate readimmediately using an excitation wavelength of 360 nm and an emissionwavelength of 460 nm. At least two each of the reference standard andsample curves were required to have an R-squared value of 0.95 to beconsidered valid.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-49. (canceled)
 50. A pharmaceutical composition comprising apolypeptide comprising the amino acid sequence: Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (aminoacids 3-60 of SEQ ID NO:2), and one or more pharmaceutically acceptablebuffers, carriers and excipients.
 51. The pharmaceutical composition ofclaim 50, wherein the polypeptide comprises the amino acid sequence: GluAla Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala AlaHis Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile TyrGly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys LysMet Cys Thr Arg Asp (SEQ ID NO:2).
 52. The pharmaceutical composition ofclaim 50, wherein the polypeptide consists of the amino acid sequence:Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr GlyGly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp (amino acids 3-60 of SEQ ID NO:2).
 53. Thepharmaceutical composition of claim 51, wherein the polypeptide consistsof the amino acid sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe ThrArg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg PheGlu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2). 54.The pharmaceutical composition of claim 50, wherein the composition is adry lyophilized powder.
 55. A container comprising the pharmaceuticalcomposition of claim
 50. 56. The container of claim 55, wherein thecontainer is an infusion bottle or bag.